Aire is a transcriptional regulator that controls immunological tolerance by inducing the expression of a large repertoire of gene transcripts encoding peripheral-tissue self-antigens in thymic medullary epithelial cells (MECs), thereby promoting the clonal deletion of thymocytes capable of recognizing these self-antigens. Mutations in the AIRE gene underlie a rare multi-organ autoimmune disease called APECED, and abnormalities in the Aire-pathway have also been implicated in certain of the more common autoimmune disorders, notably type-1 diabetes and myasthenia gravis. This project issues from our novel, very surprising, finding that Aire's impact on immunological tolerance has an intriguing temporal window: its expression during the perinatal period is necessary and sufficient to avoid the multi-organ autoimmune disease typical of Aire-knockout mice. Hence, the project's overall goal is to understand why Aire must be expressed in perinatal mice to guard against autoimmunity, but is dispensable in adults. Experiments proposed for this MERIT extension fall under the original Specific Aims: 1. To compare the repertoires of Aire-dependent MEC transcripts from adult and perinatal mice: 2. To assess the effect of Aire gene shutdown in adults versus perinates on emerging self-reactive effector T cells: and 3. To evaluate the influence of Aire turnoff in adult vs perinatal mice on the emerging repertoire of regulatory T cells. Interesting experimental follow-ups include: functional evaluation of the impact of Aire-dependent splicing on T cell tolerance, and a single-cell RNA-seq comparison of the repertoires of perinatal and adult MEC transcripts (Aim 1); elucidation of the mechanisms underlying Aire-dependent selection of T??17 cells, and study of their function(s) (Aim 2); and an exploration of both Aire's influence on and the contribution of perinatal Tregs to tissular and tumor Treg compartments. Results from these studies will not only elucidate the nature of the temporal window of Aire's impact on immunological tolerance but, more generally, should yield new insights into neonatal tolerance, which has fascinated immunologists for decades. RELEVANCE (Se instructions): This study focuses on the cellular mechanisms of Aire, the protein encoded by the gene mutated in individuals with APECED, a disease characterized by multi-organ autoimmunity. While APECED patients are rather rare, the supposition is that, as is often the case, insights into its molecular etiology will be applicable to more common diseases; indeed, genetic studies on several human autoimmune disorders, notably type-1 diabetes and myasthenia gravis, have revealed that they also, at least partially, reflect defects in the Aire-dependent pathway of immunological tolerance.